Actinobacillus suis antigens

ABSTRACT

The invention provides immunogenic compositions useful for inhibiting, treating, protecting, or preventing infection by  Actinobacillus suis . These immunogenic compositions are demonstrated to usefully stimulate immunogenic responses in treated pigs. Some vaccines stimulated reactions sufficient to be protective against  A. suis . In addition, the invention provides kits comprising the immunogenic compositions; as well as, methods of using the compositions and kits.

RELATED APPLICATIONS

This application relates to and claims priority to U.S. ProvisionalPatent Application No. 61/259,728, which was filed Nov. 10, 2009. Theteachings and contents of which are incorporated herein by reference intheir entirety. All applications are commonly owned.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to methods and compositions useful forinhibiting, treating, protecting, or preventing infection byActinobacillus suis.

B. Description of the Related Art

Actinobacillus suis has recently emerged as a new threat to the swineindustry in the United States. Previously associated with high mortalityin “high health” herds, A. suis is now recognized as an importantpathogen of conventional herds. It is particularly detrimental toyounger animals. Infection results in actinobacillosis and can causesudden death in both neonate and weaned pigs. Symptoms in weaned pigsinclude anorexia, fever, cyanosis, congestion of the extremities,respiratory distress, pneumonia, necrotizing pneumonia, persistentcough, skin lesions, and fatal septicemia. Pneumonia, arthritis,septicemic signs, pleurisy, pericarditis, and miliary abscesses areknown to occur in finishing pigs. Actinobacillosis also causes metritisand abortion in sows.

The gross pathology of actinobacillosis is characterized by lesionsfound in lungs, kidney, heart, liver, spleen, intestines and skin;hemorrhages and necrosis; and pneumonic lesions resemblingpleuropneumonia. Histopathologically, the disease is characterized bythe presence of bacterial thromboemboli with accompanyingfibrinohemorrhagic necrosis in the vessels of various tissues;necrotizing bronchopneumonia; and pleuritis. Causes and contributingfactors to infection include precipitation by Porcine Reproductive andRespiratory Syndrome (PRRS) infection, teeth clipping, de-tailing,scrubbed knees, and entry via either respiration, cuts, or abrasions.

Actinobacillus suis is an opportunistic, gram-negative, non-motile,aerobic and facultative anaerobic coccobacillus that colonizes the upperrespiratory tract. Genotyping of A. suis isolates recovered fromclinical cases in the North American swine herds has revealed a limitedgenetic variability, with only 13 strains being identified among 74isolates recovered from 29 different herds. The Simpson's diversityindex (also known as species diversity index, see Simpson, 1949) for A.suis genotypes is 0.64, meaning that a random isolate has a 64% chanceof being included in a unique genotype group using for example BOX-PCR(Simpson, 1949; Versalovic et al, 1991; Oliveira et al, 2007). Comparedwith H. parasuis, for example, which has a diversity index of 0.93(Oliveira et al, 2007), A. suis is relatively clonal.

The phenotypic diversity of A. suis is also relatively limited. Only 2serovars, namely O1 and O2 (Rullo, Papp-Szabo and Michael, 2006), andthree capsular types, K1-3, have been described so far. Pathogenicitystudies suggest that isolates from serogroup O2 tend to be more virulentthan O1 isolates (Slavic, DeLay and Hayes, 2000). Serotyping of A. suisisolates used for autogenous vaccine production also confirms that ahigher percentage of O2 isolates were associated with clinical diseasecompared with O1 isolates (Slavic, Toffner, and Monteiro, 2000).Although some of the A. suis virulence factors are known (e.g. the RTXtoxins Apx I_(var. suis) and Apx II_(var. suis)), the factors that maytrigger systemic infection still remain to be defined. Some of thesepotential factors include lipopolysaccharide (LPS) and capsularpolysaccharides (CPS), outer membrane protein A (OmpA), proteases, andiron acquisition.

Currently, there are no commercial vaccines available for the control ofA. suis, and most field veterinarians rely on autogenous vaccines andantimicrobial treatments to control disease. The development of avaccine that will potentially protect against most isolates in the fieldis desirable; however, necessary data regarding the association betweengenotype, serovar, toxin, protein profiles, and factors that areinvolved in the pathogenesis of A. suis infection still remain to bedefined. Herein, such data are provided, as well as, vaccines, and theirmethods of use, against A. suis.

SUMMARY OF THE INVENTION

The present invention provides new immunogenic compositions that areuseful for protecting a subject against Actinobacillus suis infection.These compositions are also useful for inhibiting, treating, orpreventing infection by various strains or types of Actinobacillus suis.

Compositions of the invention comprise a supernatant collected from oneor more A. suis cultures grown to between 0.650 OD₆₅₀ and 0.850 OD₆₅₀;and an adjuvant. Preferably the supernatant is inactivated, mostpreferably by formalin. The supernatant is also preferably filtered,such as through a 45 micron filter. Filtration may occur before or afterinactivation as deemed appropriate for a given situation. Thesupernatant, more preferably the filtered supernatant, is essentiallyfree from A. suis cells but may contain multiple cellular components.

The skilled artisan will recognize that any of a variety of adjuvantsmay be suitably included in a composition of the invention. Oneexemplary adjuvant is Emulsigen®-D, an oil-in-water emulsion whichincorporates dimethyldioctadecylammonium bromide (DDA). Thedetermination of adjuvant will, in part, depend upon the nature of thesubject that is to receive the composition; the method of administrationto the subject; and conditions under which the composition is to beadministered. For example, the adjuvant and supernatant, or filteredsupernatant, may be admixed together prior to administration to asubject, administered simultaneously, or administered sequentially to asubject.

Suitable subjects of the immunogenic compositions of the inventioninclude animals and humans. Animals in which the immune response isstimulated by use of compositions or methods of the invention includelivestock, such as pigs, calves, chickens, goats, and sheep, anddomestic animals, such as mice, rabbits, dogs, cats, and horses.Preferred animals include porcines, murids, equids, lagomorphs, andbovids. Most preferably the animal is a porcine.

The invention also provides methods of provoking an immune responseagainst Actinobacillus reducing the incidence of or severity of aclinical sign associated with Actinobacillus suis infection in a subjectcomprising administering to the subject an immunogenic composition ofthe invention. Clinical signs associated with A. suis that may bereduced in incidence or severity in a subject include meningitis,septicemia, metritis, pneumonia, crysipelas-like lesions, and abortion.Any one of which may be lessened by the administration of a compositionof the invention relative to a subject not receiving the immunogeniccomposition. Preferred compositions of the invention elicit a protectiveimmunological response that is at least a 10% reduction in at least oneclinical sign of an A. suis infection.

A preferred Actinobacillus suis infection that may be reduced byadministration of a composition of the invention is A. suis ISU-8594.

The invention also provides methods of making or preparing immunogeniccompositions of the invention that may be useful in the making of amedicament. Such methods include the steps of growing an Actinobacillussuis culture to between 0.650 OD₆₅₀ and 0.850 OD₆₅₀; collecting asupernatant from the culture; filtering the supernatant to yield afiltrate; and mixing the filtrate with an adjuvant. Such methods mayalso include inactivating the supernatant, preferably prior to admixingthe supernatant with an adjuvant. Inactivation may occur either beforeor after filtering the supernatant.

The invention further provides methods of diagnosing an Actinobacillussuis infection in a subject. Such methods comprise: a) providing afiltered supernatant prepared by growing an A. suis culture to between0.650 OD₆₅₀ and 0.850 OD₆₅₀, collecting supernatant from the culture,and filtering the supernatant; b) contacting the filtered supernatantwith a sample obtained from the subject; and c) identifying the subjectas having an A. suis infection if an antibody capable of binding acomponent in the filtered supernatant is detected in the sample.

Those of skill in the art will be familiar with a variety of techniquessuitable for ascertaining if an antibody is capable of binding to acomponent. For example, binding may be detected by using a secondantibody capable of binding the antibody in the sample. Binding by suchsecond antibodies may by detected by a colorimetric assay or othersuitable means.

The invention also provides kits that comprise: a) a filteredsupernatant prepared by growing an Actinobacillus suis culture tobetween 0.650 OD₆₅₀ and 0.850 OD₆₅₀, collecting a supernatant of theculture, and filtering the supernatant; b) an adjuvant; and c) acontainer for packaging the supernatant and adjuvant. The filteredsuperantant and adjuvant may be packaged together or separately.

A kit may further comprise instructions for use of the kit. It may alsocomprise a means of administering the filtered supernatant and adjuvantto a subject. A means of admixing the supernatant and adjuvant togethermay also be included in a kit.

Compositions of the invention may further comprise a veterinarilyacceptable carrier, second adjuvant, or combination thereof. Suchcompositions may be used as a vaccine and comprise an attenuatedvaccine, an inactivated vaccine, or combinations thereof. Such vaccineselicit a protective immunological response against at least one diseaseassociated with Actinobacillus.

Preferred inactivation agents for use in methods of the invention areselected from the group consisting of binary ethyleneimine (BEI) andformalin. Formalin is a more preferred inactivation agent. Those ofskill in the art will recognize that other inactivation agents andmethods (i.e. heating, changing pH, etc.) are known in the art and maybe interchangeably used in the practice of the invention, as long as,such agents or deactivation methods do not adversely alter theimmunogenic properties or safety of the composition produced.

Methods of the invention may also comprise admixing a composition of theinvention with a veterinarily acceptable carrier, adjuvant, orcombination thereof. Those of skill in the art will recognize that thechoice of carrier, adjuvant, or combination will be determined by thedelivery route, personal preference, and animal species among others.

Preferred routes of administration include intranasal, oral,intradermal, and intramuscular. Administration in drinking water, mostpreferably in a single dose, is preferred. The skilled artisan willrecognize that compositions of the invention may also be administered intwo or more doses, as well as, by other routes of administration. Forexample, such other routes include subcutaneously, intracutaneously,intravenously, intravascularly, intraarterially, intraperitnoeally,intrathecally, intratracheally, intracutaneously, intracardially,intralobally, intramedullarly, intrapulmonarily, or intravaginally.Depending on the desired duration and effectiveness of the treatment,the compositions according to the invention may be administered once orseveral times, also intermittently, for instance on a daily basis forseveral days, weeks or months and in different dosages.

The invention also provides kits for vaccinating a subject comprising aset of printed instructions; a dispenser capable of administering avaccine to an animal; and a supernatant from an A. suis culture havingone or more components that effectively stimulates an immune response ina subject. Kits of the invention may further comprise a veterinarilyacceptable carrier, adjuvant, or combination thereof.

A dispenser in a kit of the invention is capable of dispensing itscontents as droplets; and the A. suis supernatant included in the kit iscapable of reducing the severity of at least one clinical sign of an A.suis infection when administered to a subject. Preferably, the severityof a clinical sign is reduced by at least 10% as compared to anuntreated, infected subject.

An “immunogenic or immunological composition” refers to a composition ofmatter that comprises at least one A. suis supernatant, or immunogenicportion thereof, that elicits an immunological response of a cellular orantibody-mediated immune response to the composition in the subject. Ina preferred embodiment of the present invention, an immunogeniccomposition induces an immune response and, more preferably, confersprotective immunity against one or more of the clinical signs of aActinobacillus infection.

An “immune response” or “immunological response” means, but is notlimited to, the development of a cellular and/or antibody-mediatedimmune response to the composition or vaccine of interest. Usually, animmune or immunological response includes, but is not limited to, one ormore of the following effects: the production or activation ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells, directed specifically to an antigen or antigensincluded in the composition or vaccine of interest. Preferably, thevaccinated subject will display either a therapeutic or a protectiveimmunological (memory) response such that resistance to new infectionwill be enhanced and/or the clinical severity of the disease reduced.Such protection will be demonstrated by either a reduction in number ofsymptoms, severity of symptoms, or the lack of one or more of thesymptoms associated with the infection of the pathogen, a delay in theof onset of clinical symptoms, reduced pathogen persistence, a reductionin the overall pathogen load and/or a reduction of pathogen excretion.

“Protection against A. suis”, “protective immunity”, “functionalimmunity”, and similar phrases, mean an immune response against A. suisgenerated by an immunization schedule that results in fewer deleteriouseffects than would be expected in a non-immunized subject that has notbeen previously exposed to A. suis. That is, the severity of thedeleterious effects of the infection are lessened in an immunizedsubject because the subject's immune system is resistant to thebacterium. Infection may be reduced, slowed, or possibly fullyprevented, in an immunized subject, preferably a pig. Herein, wherecomplete prevention of infection is meant, it is specifically stated. Ifcomplete prevention is not stated then the term includes partialprevention.

Herein, “reduction of the incidence and/or severity of clinical signs”or “reduction of clinical symptoms” means, but is not limited to,reducing the number of infected subjects in a group, reducing oreliminating the number of subjects exhibiting clinical signs ofinfection, or reducing the severity of any clinical signs that arepresent in the subjects, in comparison to wild-type infection. Forexample, it should refer to any reduction of pathogen load, pathogenshedding, reduction in pathogen transmission, or reduction of anyclinical sign symptomatic of A. suis infection. Preferably theseclinical signs are reduced in subjects receiving the composition of thepresent invention by at least 10% in comparison to subjects notreceiving the composition and may become infected. More preferablyclinical signs are reduced in subjects receiving the composition of thepresent invention by at least 20%, preferably by at least 30%, morepreferably by at least 40%, and even more preferably by at least 50%.

The term “increased protection” herein means, but is not limited to, astatistically significant reduction of one or more clinical symptomswhich are associated with A. suis infection in a vaccinated group ofsubjects vs. a non-vaccinated control group of subjects. The term“statistically significant reduction of clinical symptoms” means, but isnot limited to, the frequency in the incidence of at least one clinicalsymptom in the vaccinated group of subjects is at least 20%, preferably30%, more preferably 50%, and even more preferably 70% lower than in thenon-vaccinated control group after the challenge with an infectiousActinobacillus bacterium.

Those of skill in the art will understand that the compositions usedherein may incorporate known injectable, physiologically acceptablesterile solutions. For preparing a ready-to-use solution for parenteralinjection or infusion, aqueous isotonic solutions, e.g. saline or plasmaprotein solutions, are readily available. In addition, the immunogenicand vaccine compositions of the present invention can includeveterinary-acceptable carriers, diluents, isotonic agents, stabilizers,or adjuvants.

As used herein, “a veterinary-acceptable carrier” includes any and allsolvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and the like. In some preferredembodiments, and especially those that include lyophilized immunogeniccompositions, stabilizing agents for use in the present inventioninclude stabilizers for lyophilization or freeze-drying.

In some embodiments, the immunogenic composition of the presentinvention contains an adjuvant. “Adjuvants” as used herein, can includealuminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion,oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion canbe based in particular on light liquid paraffin oil (EuropeanPharmacopea type); isoprenoid oil such as squalane or squalene; oilresulting from the oligomerization of alkenes, in particular ofisobutene or decene; esters of acids or of alcohols containing a linearalkyl group, more particularly plant oils, ethyl oleate, propyleneglycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) orpropylene glycol dioleate; esters of branched fatty acids or alcohols,in particular isostearic acid esters. The oil is used in combinationwith emulsifiers to form the emulsion. The emulsifiers are preferablynonionic surfactants, in particular esters of sorbitan, of mannide (e.g.anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycoland of oleic, isostearic, ricinoleic or hydroxystearic acid, which areoptionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymerblocks, in particular the Pluronic products, especially L121. See Hunteret al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) andTodd et al., Vaccine 15:564-570 (1997).

Other exemplary adjuvants are the SPT emulsion described on page 147 of“Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powelland M. Newman, Plenum Press, 1995, and the emulsion MF59 described onpage 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among then, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCabopol 971P. Among the copolymers of maleic anhydride and alkenylderivative, are the copolymers EMA (Monsanto), which are copolymers ofmaleic anhydride and ethylene. The dissolution of these polymers inwater leads to an acid solution that will be neutralized, preferably tophysiological pH, in order to give the adjuvant solution into which theimmunogenic, immunological or vaccine composition itself will beincorporated.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, ornaturally occurring or recombinant cytokines or analogs thereof orstimulants of endogenous cytokine release, among many others.

“Diluents” can include water, saline, dextrose, ethanol, glycerol, andthe like. Isotonic agents can include sodium chloride, dextrose,mannitol, sorbitol, and lactose, among others. Stabilizers includealbumin and alkali salts of ethylendiamintetracetic acid, among others.

It is expected that an adjuvant can be added in an amount of about 100μg to about 10 mg per dose, preferably in an amount of about 100 μg toabout 10 mg per dose, more preferably in an amount of about 500 μg toabout 5 mg per dose, even more preferably in an amount of about 750 μgto about 2.5 mg per dose, and most preferably in an amount of about 1 mgper dose. Alternatively, the adjuvant may be at a concentration of about0.01 to 50%, preferably at a concentration of about 2% to 30%, morepreferably at a concentration of about 5% to 25%, still more preferablyat a concentration of about 7% to 22%, and most preferably at aconcentration of 10% to 20% by volume of the final product.

Herein, “effective dose” means, but is not limited to, an amount of A.suis supernatant or filtered supernatant that elicits, or is able toelicit, an immune response that yields a reduction of clinical symptomsin a subject to which the supernatent is administered.

“Safety” refers to the absence of adverse consequences in a vaccinatedsubject following vaccination, including but not limited to: potentialreversion of a bacterium-based vaccine to virulence, clinicallysignificant side effects such as persistent, systemic illness orunacceptable inflammation at the site of vaccine administration.

The terms “vaccination” or “vaccinating” or variants thereof, as usedherein means, but is not limited to, a process which includes theadministration of a composition of the invention that, when administeredto a subject, elicits, or is able to elicit—directly or indirectly—animmune response in the subject against A. suis.

Methods for the treatment or prophylaxis of infections caused by A. suisare also disclosed. The method comprises administering an effectiveamount of the immunogenic composition of the present invention to asubject, wherein said treatment or prophylaxis is selected from thegroup consisting of reducing signs of A. suis infection, reducing theseverity of or incidence of clinical signs of A. suis infection,reducing the mortality of subjects from A. suis infection, andcombinations thereof.

“Mortality”, in the context of the present invention, refers to deathcaused by A. suis infection, and includes the situation where theinfection is so severe that an animal is euthanized to prevent sufferingand provide a humane ending to their life.

“Attenuation” means reducing the virulence of a pathogen. In the presentinvention “attenuation” is synonymous with “avirulent”. In the presentinvention, an attenuated bacterium is one in which the virulence hasbeen reduced so that it does not cause clinical signs of an A. suisinfection but is capable of inducing an immune response in the targetsubject, but may also mean that the clinical signs are reduced inincidence or severity in subjects infected with the attenuated A. suisin comparison with a “control group” of subjects infected withnon-attenuated A. suis and not receiving the attenuated bacterium. Inthis context, the term “reduce/reduced” means a reduction of at least10%, preferably 25%, even more preferably 50%, still more preferably60%, even more preferably 70%, still more preferably 80%, even morepreferably 90% and most preferably of 100% as compared to the controlgroup as defined above.

An “effective amount” for purposes of the present invention, means anamount of an immunogenic composition capable of inducing an immuneresponse that reduces the incidence of or lessens the severity of A.suis infection in a subject. An effective amount refers to colonyforming units (CFU) per dose or logs/dose.

“Long-lasting protection” shall refer to “improved efficacy” thatpersists for at least 3 weeks, but more preferably at least 3 months,still more preferably at least 6 months. It is most preferred that thelong lasting protection shall persist until the average age at whichporcine animals are marketed for meat.

Herein, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. In thisapplication, the use of “or” means “and/or” unless stated otherwise.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Daily average temperatures among treatment groups from day 0 today 41 of the study.

DETAILED DESCRIPTION

The invention provides methods and compositions useful for inhibiting,treating, protecting, or preventing infection by Actinobacillus suis.Herein are described the effectiveness of A. suis prototype vaccinesagainst a heterologous challenge of various serotypes or isolates of A.suis. These vaccines were demonstrated to usefully stimulate immunogenicreactions in treated animals. Some vaccines stimulated reactionssufficient to be protective against A. suis.

The A. suis vaccine prototypes were comprised of different, culturefractions (e.g. whole cell, supernatant, or outer membrane protein(omp)) and a Ingelvac® APP-ALC live vaccine against A. pleuropneumoniae(APP). The whole cell vaccine was made from the pelleted material thatresulted from a centrifugal spin of a cell culture. The culturalsupernatant was made from the supernatant that resulted from thecentrifugal spin, and may have included exotoxins, endotoxins, secretedor sloughed-off omps, and other cellular components that were notremoved during the spin (see Example 1). The Ingelvac® APP-ALC livevaccine was included because APP encodes Apx I and II toxins that arevery similar to A. suis toxins. It was hypothesized that the Ingelvac®APP-ALC vaccine may provide some cross protection against A. suis.

This efficacy study consisted of six treatment groups of weaned 3week-old ±7 days of age pigs. Treatment group 1 (15 pigs) received a 1×2mL intramuscular (IM) dose of a formalin-inactivated, whole cellfraction of A. suis on days 0 and 21 of the study, respectively.Treatment group 2 (15 pigs) received a 1×2 mL IM dose of aformalin-inactivated A. suis culture supernatant fraction on days 0 and21 of the study, respectively. Treatment group 3 (15 pigs) received a1×2 mL IM dose of an A. suis outer membrane protein (omp) cellularfraction on days 0 and 21 respectively. Treatment group 4 (15 pigs)received a 1×2 mL IM dose of Ingelvac® APP-ALC on days 0 and 21respectively. Treatment group 5 (15 pigs) designated as “challengecontrols” received a 1×2 mL IM dose of placebo on days 0 and 21respectively. Treatment group 6 (10 pigs) were designated “strictcontrols” and did not receive vaccine or placebo treatment.

On the day of challenge (day 35) the pigs in groups 1-5 received a 6 mLdose/pig, 3 mL applied to each nostril, of A. suis strain ISU-8594 viaintranasal (IN) inoculation containing 1×10^(9.0) logs/dose. Generalobservations were monitored throughout the study, days 0 to 41, torecord the overall health of the pigs, as well as, injection sitereactivity and rectal temperatures. On Day 41 of the study, all animalswere euthanized, and all sections of the lung were scored fordetermining the percentage of lung pathology. Fresh and fixed (lungsonly) samples of the lung (3 lobes with lesions, if present), liver,kidney, spleen, tonsil, and swabs of nasal turbinates, trachea, bronchi,meninges, and heart blood were collected from each animal and werecultured for bacterial isolation and histopathology (lungs only).

The A. suis fractions and placebo groups were adjuvanted withEmulsigen®-D, and a few adverse injection site reactions were noted inthese groups. One pig in the supernatant group (group 2) was noted witha mild reaction at the injection site during the first vaccinationevent. Overall, the Emulsigen®-D in combination with the test articlesdid not produce any significant injection site reactions in thesupernatant and omp groups. However, the whole cell group did have a fewanimals with abscesses present at the time of necropsy, which could havebeen due to the test article formulation that consisted of aconcentrated whole cell stock. The administration of these vaccineprototypes (i.e. whole cell, supernatant, and omp) appeared to bewell-tolerated during the time of administration. The APP-ALC group hadsome injection site swelling, which is consistent with the productliterature that notes swelling in 11% of the vaccinated animals.

The general overall health of the pigs in each of the treatment groupswas good during the pre-challenge period. Only one pig, from treatmentgroup 4, was removed from the study during this period. This pig wasremoved due to a bacterial infection believed not to be associated withadministration of the APP vaccine. Further observations showed one pigin the challenge control group was lame on its right leg from day 7 today 24 of the study. This pig skewed the clinical observation resultsduring the pre-challenge period and could have been considered anoutlier and removed from the study data analysis. Also, one pig showedpoor body condition in the whole cell group and died after a few days.None of the pigs in the pre-vaccination period showed signs ofrespiratory difficulties.

Assessment of the primary and secondary parameters were validated bysignificant (p<0.05) increases in lesion development that occurred inthe challenge control group (treatment group 5) compared to the strictcontrol group (treatment group 6). Results of this assessment validatedthe virulent pure culture A. suis challenge model in pigs. Furthercomparisons were made against the challenge control and not to thestrict control group when comparing groups 1-4.

During the six day challenge period one pig died of acute death due toA. suis infection one day post-challenge in the whole cell group. Basedon previous challenge studies, the intranasal route and the potency wereappropriate to cause significant lesion development in the test animalsand apparent fatal sudden death, as was the case for this one pig in thewhole cell group.

Based on average lung lesion scores and percent with a lesion, grosslung lesion scores appeared to be most severe in the challenge group.Statistical analyses showed no statistical differences (p<0.05) inaverage lung lesion scores or percent with a lesion present whencomparing groups 1 through 5. Numerically the challenge controlsreceived overall the highest average gross lung lesion scores followedby the whole cell and omp groups, which also received relatively highscores. Lesion development was lower in the supernatant and APP groups,which received lower numerical scores compared to the other groups. Allgroups 1-5 had at least 50% of the pigs with a gross lesion score, andthe challenge and omp groups had as much as 80% of the pigs with lesiondevelopment.

Furthermore, microscopic lung lesion analysis was based on the severityof the hallmark traits of an A. suis infection, i.e. bronchopneumonia,necrosis, and pleuritis. Bronchopneumonia and necrosis were more severein the challenge control and omp groups where microscopic lesion scoreswere lower than in the supernatant and APP groups. The APP group showedthe best signs of protection against an A. suis infection based on themicroscopic lesion data with statistical differences (p<0.05) foundbetween group 3 (omp) for necrosis and pleuritis and the group 5(challenge) for pleuritis. Presence of bacterial colonies in all of thegroups ranged from about 20% to about 46.7% with the lowest percentcolonization occurring in the whole cell and APP (20.0%) groups.

The secondary parameters monitored were used to support the primaryefficacy parameters (i.e., gross and microscopic (IHC) lesions)responses due to vaccine and challenge for each treatment group.Clinical observations made during the challenge period indicated thatthe challenge controls had an overall higher score when compared to thevaccine treatment groups 1-4, showing that the challenge controlsresponded to the challenge. The supernatant group did not seem to be asclinically affected. However, all groups did have some pigs that didhave some respiratory issues with a cough, body condition, or behavior.

The treated groups saw an increase in average rectal temperatures, above104° F., four hours after the first vaccination event. Temperatures forthe APP and omp groups spiked at or above 104.9° F., which was thecut-off of pyrexia following four hours post vaccination, and lastedabout twenty-four hours for the omp group. Temperatures in the groupswere returning back to normal by day 1 or 3. For the booster event,temperatures were only taken on day 28 and not at 4 hrs or on days 1, 3,and 5 post-vaccination as with the first vaccination. During the boosterperiod, on day 28 the APP group was statistically different (p<0.05)when compared to the whole cell and supernatant groups. During thechallenge period, the challenge controls had an overall higher rectaltemperature at both the beginning and end of the period. The whole cellgroup had the highest rectal temperatures during days 38 and 39. Thechallenge control group had the highest overall percent animals febrile( 10/15) followed by the whole cell ( 8/15), OMP ( 5/15), supernatant (2/15), and APP ( 1/15) groups, respectively.

Bacterial isolation was highest in the lung and tonsil samples. Thehighest recovery of A. suis occurred in the challenge control group. Thestrict control group was negative for A. suis except in nasal and tonsilsamples, which had 3 out of 10 and 1 out of 10 respectively. Theseresults were recorded as a positive and not verified by BCA or othermeans. Possible reasons for these results could have been an inadvertentmisread of the agar plate or a cross-contamination event during theprocessing of the swabs. Furthermore, three parameters had very lowrecovery rates of A. suis which were the spleen, meninges, and heartblood, and thus, were not included in the analysis. Overall the rate ofsepticemia was low due to low recovery of A. suis in the visceral organsand heart blood. There was also evidence that this challenge crossed theblood brain barrier in three of the animals that were severely infected.Other detected bacteria were Bordetella, alpha and beta streptococci.

Serology testing using a western blot procedure was performed to testfor reactivity against the test article that was used to vaccinate thetreatment groups. The western blots showed detectible seroconversion tothe whole cell, supernatant, and omp fractions at 1:100 dilution. TheAPP fraction had the best reactivity and was detectable at a dilution of1:1000, confirming that the host recognized some part of the fractionsused for vaccination of each treatment group.

Overall the best protection occurred in the pigs vaccinated with the APPand supernatant prototype vaccines. Comparatively, neither the wholecell nor omp vaccines or LPS derived antigens provided much protectionfrom A. suis challenge. Details of the challenge study are provided inthe Examples below.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs at the time of filing. Ifspecifically defined, then the definition provided herein takesprecedent over any dictionary or extrinsic definition. Further, unlessotherwise required by context, singular terms shall include pluralities,and plural terms shall include the singular. Herein, the use of “or”means “and/or” unless stated otherwise. All patents and publicationsreferred to herein are incorporated by reference.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Prototype Vaccines and Challenge Treatment

A. Preparation of Vaccine Prototypes

Growth of A. suis (KSU-1) Cultures:

One milliliter of A. suis (103007-1KSU) lot #201-9,10,11 was used toinoculate 150 ml of BHI media (lot #195-121) in a 250 ml spinner. Theinoculated media was placed at 37° C. at about 72 rpm for six hours.Afterwards, about 2.5 ml of the inoculated media was added to about 2500ml fresh BHI media. This step was repeated until four batches of 2500 mlwere prepared. The four batches were incubated at 37° C. overnight atabout 72 rpm. Two batches were used to isolate whole cell OMP, andsupernatant fractions, one batch was used to prepare a concentratedwhole cell fraction, and the final batch was used for LPS extraction.

Harvesting of Cell Fractions:

Optical densities of batches were read fifteen hours after inoculation.Optical densities ranged from 0.775 to 0.815 at 600 nm. BAPs werestreaked from each culture to verify that the cultures were pure for A.suis.

To collect the OMP and supernatant fractions, two culture batches weretransferred to 500 ml centrifuge tubes, placed in a JA-10 rotor, andspun at 10K for 10 minutes. The supernatants were decanted, and thepellets were transferred into sterile 250 mL Corning centrifuge tubes.The pellets were centrifuged at about 5000 rpm in a JA-10 rotor for 10minutes, and the remaining supernatants were removed. The pellets werepooled and placed at −80° C. until sarcosyl extraction (see below). Thedecanted supernatants were pooled, and filtered through 0.45 μm filters(Nalgene 0.45 SFCA) into sterile 2 L Pyrex bottles. The filteredsupernatant was aliquoted into 2×850 cm RBs, 81×5 mL vials, and 4×250 mLNalgene bottles. Filtered supernatant was stored at −20° C. until use.

To collect the concentrated (10×) whole cell pellet fraction, oneculture batch was harvested as above, but the pellets were resuspendedin 300 mL TSP buffer. The solution was mixed well and aliquoted into50×2 mL vials and 4×50 mL vaccine bottles. Resuspended whole cellpellets were stored at −80° C. until use.

To prepare the LPS fraction, A. suis culture was harvested as describedabove. The pellets were resuspended in 1×PBS buffer and re-centrifugedat 10K rpm for ten minutes. Supernatants were decanted, and the pelletswere resuspended in 1 L of 1×PBS buffer. An optical density reading atabout 600 nm was 1.003, which is within the expected range of 0.8-1.200for LPS extraction. The LPS fraction was aliquoted into 3×250 mL Nalgenebottles and stored at −80° C. until use. The remaining volume of LPSfraction was stored at 4° C. for LPS extraction.

KSU-1 Whole Cell Formalin-Inactivated Vaccine:

The optical density (OD) of the cell culture measured at 600 nanometerwavelength was at 0.775 at the time of harvest. In previous studies,this optical density has correlated to approximately 8.7 logs/mL basedon the logarithmic estimate curve to determine logs/mL based on colonyforming units (cfu). The whole cell fraction was concentrated 10× andfrozen. The frozen stock was then formalin-inactivated, and diluted (54mLs of culture into 26 mLs of 1× phosphate buffered saline (pbs), to67.5% of the total fraction. The inactivated, diluted vaccine stock wasthen stored at 4° C.

The whole cell formalin-inactivated vaccine stock was removed from 4° C.for formulation. Working in a bio-safety hood 80 mLs of the material wasaliquoted out into a sterile 100 mL Pyrex bottle containing a magneticstir bar. The bottle was placed on a stir plate, and with continuousstifling, 20 mLs (20% v/v) of the adjuvant (Emulsigen®-D) was added over1 minute. The adjuvanted prototype was then stirred for an additional 10minutes. The material was transferred to a 100 mL vaccine bottle whereit was capped and placed at 4° C. Lot nos. for the whole cellformalin-inactivated vaccine prototype were N201-110-WC-073108 (day 7)and N201-122-WC-082108 (day 21).

Sterility was verified by no growth on 5% Sheep Blood Agar Plate (BAP)after 28 hrs and 30 hrs of 37° C. aerobic incubation for day 0 and day21 respectively. Vaccine prototype material was kept on ice prior toadministration to the test animals and during the entire vaccineadministration procedure.

KSU-1 Supernatant Formalin-Inactivated Vaccine:

The supernatant prototype was formulated from the 10× concentration stepof the whole cell fraction preparation (see above) by collecting thesupernatant and filtering it through a 0.45 micro filter. The resultingfiltered supernatant was frozen. This supernatant was then formalininactived and stored at 4° C. until formulated. The formulation step wasas for the whole cell formalin-inactivated vaccine. Lot nos. for thesupernatant formalin-inactivated vaccine prototype wereN201-110-Supe-073108 (day 7) and N201-122-Supe-082108 (day 21).

Sterility was verified, and the vaccine prototype material wasmaintained prior to administration to the test animals and during theentire vaccine administration procedure as previously described.

KSU-1 OMP Fraction Vaccine:

The OMP fraction was produced from pellets prepared from a KSU-1 culture(isolate BI-103007-1KSU) and stored at −80° C. by using the followingOMP sarcosyl extraction procedure. Pellets were thawed on the benchtop.The pellets were resuspended (22.5 g wet weight) with 130 mL of 10 mMHEPES balanced salt solution and aliquoted into four 50 mL conicalFalcon tubes (40 mL/tube). (A 1 mL resuspended pellet sample wasretained for protein quantification and analysis.) The resuspendedpellets were placed in an ice bath and sonicated for 1 minute using(Large/Microtip) probe (50 mL aliquots) with a sonication pulse settingof 1 pulse/sec. The sonication was repeated three times. (A 1 mLpost-sonication sample was retained.) The sonicated pellets werecentrifuged at 17,000×g for 20 min in 50 mL tubes. Afterwards, thesupernatants were decanted and pooled into a separate container. Pelletswere stored at 4° C. until after a bicinchoninic acid assay (BCA) andthen discarded.

The pooled supernatants were run in an ultra centrifuge, ˜0.0128kg/tube, at a setting of 124,000×g (30,000 rpm), 1:10 hours, 4° C., withmaximum acceleration, and no brake. Supernatants were decanted, pooledwith the earlier supernatant, and stored at 4° C. About 1 mL of 10 mMHEPES was added on top of each pellet in the ultra centrifuge tubes, andthe pellets were incubated overnight at 4° C. to loosen the pellets fromthe tubes. Another 2 mL of 10 mM HEPES was added to each tube toresuspend the pellets. Each resuspended pellet solution was q.s. to 6 mL(total vol.) with 10 mM HEPES, and 6 mL of 2% Sarcosyl was added toeach. Solutions were incubated for 30 min at room temperature.Afterwards, the tubes were balanced in ultra centrifuge buckets (˜0.0128kg/tube) using 2% sarcosyl and run in an ultra centrifuge at 124,000×g,1:10 hour, 4° C., with maximum acceleration, and no brake. Thesupernatants were collected into a vessel, and 1 mL of 10 mM HEPES wasadded to each pellet. The OMP pellets were stored are 4° C. for 1 hourprior to measuring protein concentration or visualization of proteins.

Duplicate samples (1:10 dilution) were run on either a reduced MOPS10-12% Bis-Tris SDS PAGE gel or a NuPAGE 4-12% Bis-Tris gel to visualizeprotein bands. Total protein concentrations of the final OMP stock weredetermined using the BCA and were determined prior to formulation withadjuvant. Based on the values obtained by BCA, the total mass of proteincontained in the OMP extraction stock was 1.75 μg/mL. Based on thisvalue, the potency of the formulated vaccine OMP prototype was 250μg/dose. The resuspended, extracted OMP fraction was aliquoted into 200μl samples, labelled, and frozen at −70° C.

Five vials containing 0.5 mLs of the extracted OMP fraction were removedfrom −70° C. These vials were thawed at room temperature. Working in abio-safety hood 23.25 mLs of 1× phosphate buffered saline (pbs) and 2.35mLs of the OMP material were aliquoted into a sterile 100 mL Pyrexbottle containing a magnetic stir bar. The bottle was placed on a stirplate, and with continuous stirring, 6.4 mLs (20% v/v) of the adjuvant(Emulsigen®-D) were added over 1 minute. The adjuvanted OMP prototypevaccine was then stirred for an additional 10 minutes. The OMP prototypevaccine was transferred to a 60 mL vaccine bottle where it was cappedand placed at 4° C. Lot nos. for the OMP vaccine prototype wereN201-110-OMP-073108 (day 7) and N201-122-OMP-082108 (day 21).

Sterility was verified, and the vaccine prototype material wasmaintained prior to administration to the test animals and during theentire vaccine administration procedure as previously described.

Ingelvac® APP-ALC Vaccine:

One bottle of APP-ALC was removed from −70° C. and thawed in luke warmwater. Once thawed, a sample was removed using a 16 gauge sterile needleand placed in a vaccine bottle for potency testing. The vaccine bottlewas placed at 4° C. Titrations of Ingelvac APP-ALC were performed onDays 0 and 21 for determining colony forming units (CFU) per dose. Thesamples were serially diluted out 10-fold and plated onto Mueller HintonChocolate Agar plates. Three reps were performed and allowed to incubateat 37° C. for 24 hrs before determining the CFU. Colony forming unitswere respectively at Day 0: 9.99 logs/dose and at Day 21: 10.2logs/dose. Lot nos. for the Ingelvac® APP-ALC vaccine wereN201-110-APP-073108 (day 0) and N201-122-APP-082108 (day 21).

Vaccine prototype material was maintained prior to administration to thetest animals and during the entire vaccine administration procedure aspreviously described.

Placebo Vaccine

Working in a bio-safety hood, 60 mLs of 1× phosphate buffered saline(pbs) was aliquoted out into a sterile 100 mL Pyrex bottle containing amagnetic stir bar. The bottle was placed on a stir plate, and withcontinuous stirring, 15 mLs (20% v/v) of the adjuvant (Emulsigen®-D) wasadded over 1 minute. The adjuvanted prototype was then stirred for anadditional 10 minutes. The material was transferred to 100 mL vaccinebottle where it was capped and placed at 4° C. The Lot no. for theplacebo vaccine was N 201-109.

Sterility was verified, and the placebo prototype material wasmaintained prior to administration to the test animals and during theentire vaccine administration procedure as previously described.

B. Preparation of Challenge Treatment

The challenge strain used was A. suis Isolate ISU-8594 p5. The challengematerial was produced by inoculating a 1 liter Belco spinner flaskcontaining 800 mLs of Brain Heart Infusion Porcine (BHI-Porcine) mediawith 5 mLs of ISU-8594 p4. The spinner was placed into a 37° C.incubator at ˜100 rpm. The culture was grown to an optical density(OD600) of 0.109—about 5 hrs and 30 minutes post inoculation. Theculture was then transferred to 6×100 mL vaccine bottles containing 100mLs total volume that were stoppered and capped. Five of the bottleswere placed into a cooler containing ice packs and one bottle whichserved to represent the other five bottles for titrations was placed inanother cooler containing ice packs. The titer of the challenge materialwas determined by CFU count generated from plating serial 10-folddilutions onto 5% sheep blood agar plates. The challenge titer obtainedwas 1.69×10⁸ cfu(s)/mL or 9.0 logs/6 mL dose. The Lot no. for thechallenge material was N201-138.

Challenge material was kept on ice prior to administration to the testanimals and during the entire challenge procedure.

Example 2 Vaccination of Pigs

A mixture of female and neutered male porcine animals of a commercialcross and 3 weeks ±7 days of age were obtained from Wilson Prairie ViewFarms. Animals were healthy with no evidence of clinical respiratorydisease and negative for bacterial respiratory pathogens such as A.pleuropneumonia, H. parasuis, S. suis, P. multocida, B. bronchiseptica,E. rhusiopathiae and A. suis. While allowance was made for animals to beexcluded from the study if and when health problems unrelated tovaccination or challenge became apparent, no animals were removed.

At the time of arrival to the test site, all animals received a 1 mLshot of Excenel® (a short acting antibiotic). The animals were housed atthe test site until the study was terminated. The animals wereear-tagged upon arrival, and housed appropriately for species, age,size, and condition. Challenge and strict control pigs were housedtogether in separate pens in a separate building away from the othertreatment groups. At the time of challenge, the challenge controls weremoved into the same building as the treated groups. All treatment groupswere housed in separate pens throughout the study. The animals wereprovided with a ration that was free of antibiotics and appropriate forspecies, age, size, and condition of the animals. The animals were ingood health and nutritional status at initiation of the study. A healthexamination was conducted by a clinical veterinarian according togenerally accepted veterinary practice on each animal prior to inclusionin the study.

A computer random number generator (Microsoft Office Excel) was used toassign each animal a unique random number. The random number was thensorted into ascending order. Assignments occurred by allocating theexperimental units across treatment groups starting with the lowestblock of random numbers and assigning them to treatment groups 1 through6. Increasing in random number, the next block of experimental unitswere then assigned to treatment groups and so on until all animals wereassigned to a treatment group.

This efficacy study consisted of 6 treatment groups of weaned 3 week-old±7 days of age pigs. Treatment group 1 (15 pigs) received a 1×2 mLintramuscular (IM) dose of formalin-inactivated whole cell A. suis ondays 0 and 21 of the study, respectively. Treatment group 2 (15 pigs)received a 1×2 mL IM dose of formalin-inactivated A. suis culturesupernatant on days 0 and 21 of the study, respectively. Treatment group3 (15 pigs) received a 1×2 mL IM dose of A. suis outer membrane protein(omp) on days 0 and 21 respectively. Treatment group 4 (15 pigs)received a 1×2 mL IM dose of Ingelvac® APP-ALC on days 0 and 21respectively. Treatment group 5 (15 pigs) designated as “challengecontrols” received a 1×2 mL dose of placebo by IM route ofadministration on days 0 and 21 respectively. Treatment group 6 (10pigs) were designated “strict controls” and did not receive vaccine orplacebo treatment. All groups were observed for 41 days.

On Day 35 of the study, Groups 1-5 received a 6 mL dose/pig (3 mLapplied to each nostril per pig) of A. suis strain ISU-8594 viaintranasal (IN) inoculation containing 1×10^(9.0) logs/dose. Group 6,the “strict control” group, did not receive any treatment or challenge.

Rectal temperatures were collected from all animals prior to treatmenton Day 0; 4 hours post inoculation; and on 1 day(s) post inoculation(DPI), 3, 5, 7, 14, 21, 28, 34, 35, 36, 37, 38, 39, 40 and 41 DPI.

Venous whole blood (6-10 mL) was collected from each animal prior totreatment on Day 0 and weekly (Days 7, 14, 21, 28, 34 and 41) for futureserological testing.

Injection sites were examined prior to treatment and at 4, 24, 48, and72 hours post inoculation in all pigs of Groups 1-5. The injection siteswere examined for swelling, hardness, and size. The data weredocumented. The following scoring system was used for injection siteobservations: swelling (0=none, 1=swelling present), appearance(0=normal, 1=hard, 2=soft, 3=abscessed, 4=draining), size (0=normal, forall others, the length by width dimensions in centimeters (i.e.length×width×diameter) were recorded).

Clinical observations were performed daily from Day 0 to Day 41 for anysigns of respiratory distress including labored breathing, sneezing,coughing, altered respiratory movements, anorexia, lameness, swelling ofjoints, dehydration, ability to stand, paddling, moribund for 2 or moreconsecutive days, or death.

Animals that had severe clinical symptoms during the study were humanelyeuthanized and necropsied to determine the cause of death. On Day 41 ofthe study, all animals were euthanized and all sections of the lung werescored for determining the percentage of lung pathology. Fresh and fixed(lungs only) samples of the lung (3 lobes with lesions, if present),liver, kidney, spleen, tonsil, and swabs of nasal turbinates, trachea,bronchi, meninges, and heart blood were collected from each animal andcultured for bacterial isolation and histopathology (lungs only).

Example 3 Evaluating Efficacy of Prototype Vaccines

A. Statistical Analysis

Statistical Analysis was performed using SAS version 9.1.3 for datamanagement and analysis. Summary statistics including mean, standarddeviation, standard error, median, range, 95% confidence intervals,coefficient of variation, and frequency distributions were generated forall data where appropriate. Analyses included all pairwise comparisonsbetween all piglet treatment groups. All tests for significance weretwo-tailed with a p≦0.05 level to determine differences betweentreatment groups.

Primary Efficacy Parameters were non-normally distributed microscopiclesion scores and gross lesion scores that were compared by WilcoxonTwo-Sample Test and Fisher's Exact Test. Secondary Efficacy Parameterswere (1) clinical signs that were compared using the Wilcoxon Two-SampleTest; (2) pyrexia that was compared using the Fisher's Exact Test andANOVA; and (3) bacterial isolation was compared using the Fisher's ExactTest.

B. Moribund Animals

Two animals were removed from the study after study initiation. AnimalID #31 (treatment group 4) was found dead on day 11 of the study. Grossexamination showed that this pig was in poor condition and thin. Itslungs had red and purple discoloration, and its abdomen had purulentexudates with fibrous peritonitis. The presumptive diagnosis was HPS ora streptococcal infection. Bacteriology cultures were performed on thefresh tissue and swab samples using standard techniques accepted in thefield. The recovered bacterial colonies were analyzed, and the analysisfound that the pig was infected with Arcanobacterium pyrogenes,Aeromonas hydrophilia, and Citrobacter freundii. Histopathology on fixedlung samples indicated no evidence of pneumonia, necrosis, pleuritis, orbacterial colonies present.

Animal ID #32 (treatment group 1) was found dead on day 36 one daypost-challenge. Gross examination showed that this pig's lungs hadsevere fibrinouses and fluid in the chest cavity. Its lungs hadconsolidation and large hemorrhagic areas. The presumptive diagnosis wasacute death due to A. suis challenge. Bacteriology cultures wereperformed on the fresh tissue and swab samples. Actinobacillus suis wasrecovered in all tissue and swab samples except the spleen and trachea.Histopathology on fixed lung samples indicated evidence of severepneumonia, necrosis, pleuritis, and bacterial colonies present.

C. General Observations

Animals were observed daily from vaccination/placebo administration tochallenge (days 0 through 35) for adverse events attributed to treatmentwith test/control articles or other non-treatment derived healthabnormalities.

General health observations for pigs in challenge control and strictcontrol groups (groups 5 and 6) were as follows: Pig #71 (challengecontrol) started showing signs of lameness on its right front on day 7and persisted to day 24 before symptoms ceased. The rest of the pigswere normal for this period of observation.

General health observations for pigs in vaccine treatment groups (groups1-4) were as follows: Pig ID #51 (whole cell, group 1) showed poor bodycondition on day 1 and persisted for three days. In treatment group 4(APP, group 4) Pig ID #31 was removed from the study as stated above.The remaining pigs in groups 1 through 4 had normal health observations.

Injection site reactions for IM-vaccinated treatment groups 1, 2, 3 and4 were as follows: Pigs #35 (supernatant, group 2) and #10 (APP, group4) had swelling of a 1×1 cm² area recorded on days 1, 2, and 3 of thestudy. No injection site evaluations were recorded for the day 21vaccination event. Clinical observations revealed swelling in the neckin pig #10 (APP, group 4) on days 4 and 5, pig #20 (APP, group 4) ondays 26 and 29, pig #40 (APP, group 4) on days 19 through 35, pig #41(APP, group 4) on days 26 through 35, and pig #46 (APP, group 4) on days14 through 35. All remaining animals had a normal, healthy dispositionduring this observation period.

At time of necropsy, pigs #69 and #75 both in treatment group 1 (wholecell) were recorded as having neck abscesses at the site of injections.No other adverse injection site recordings were reported duringnecropsy.

D. Primary Efficacy Parameters

1. Gross Lesions

At necropsy (day 41 of the study), the lungs were removed from each pigand examined for gross lesions. Individual lobes were scored for percentinvolvement, and a total score was assigned to each individual pig.Table 1 shows the average lung lesion scores for each treatment groupand the number of animals with a positive score per group.

TABLE 1 Average gross lung lesion scores by treatment group and numberof animals with a positive gross score within groups. Percent with GroupTreatment N Mean Lesion Present 1 Whole Cell 15 26.73 66.67% (10/15) 2Supernatant 15 12.68 60.00%  (9/15) 3 OMP 15 21.69 80.00% (12/15) 4 APP14 16.25 50.00%  (7/14) 5 Challenge 15 28.67^(a) 86.67^(a) (13/15) 6Strict 10 0.00^(a)  0.00^(a)  (0/10) ^(a)Groups 5 vs. 6 comparisons arestatistically significant different (p < 0.05, Wilcoxon Two Sample Testand Fishers Exact Test where appropriate).

Using the Wilcoxon Two-Sample Test and Fishers Exact Test on the numberof positive lesions per group, statistical analyses were run to comparethe means among treatment groups 5 vs. 6 and groups 1-4 vs. 5 on averagegross lesion scores. Treatment group 6 (strict controls) was negativefor gross lesion development (0) and the number of percent positivescores (0%). Evaluation of groups 1-5 for average lung lesion scores andpercent with a lesion ranged from 12.68 to 28.67 out of a possible 100and from 50.00% to 86.67% respectively.

Treatment group 5 (challenge), received the highest average gross lunglesion score (28.67) and percent with a lung lesion score (86.67%)compared to the other treatment groups. Treatment group 5 wassignificantly different (p≦0.05) from treatment group 6, when comparingaverage gross lung lesion scores and percent positive with a lesion.Treatment group 2 (supernatant) received the lowest lesion score(12.68), but had a percent lesion score that was the second lowest(60.00%) compared to treatment group 4 (APP) which had the lowestpercent lesion score (50.00%). Comparisons of groups 4 vs. 5 for percentwith a lung lesion score was almost statistically different (p=0.0502).Furthermore, treatment group comparisons for gross lesion score ofgroups 4 vs. 5 and groups 2 vs. 5 were not statistically different,(p≦0.0840) and (p≦0.0685) respectively. Numerically, treatment group 1(whole cell) had the second highest average lung lesion score of 26.73followed by the treatment group 3 (omp) with a score of 21.69. Treatmentgroup 3 (omp) had the second highest number of percent lesions presentwith 80.00% followed by treatment group 1 (whole cell) with 66.67%.

2. Microscopic Lesions

Lung sections were collected, fixed, and submitted to the Iowa StateVeterinary Diagnostic Laboratory for evaluation of non-specificmicroscopic lesion development (i.e. pneumonia, necrosis, and pleuritis)and presence of bacterial colonies. Scores were based on a nominal scaleof 1 to 3 with 1 being mild, 2 being moderate, and 3 being severe.Groups were compared based on average lesion severity and presence ofbacterial colonies (bugs) present within each tissue, and the frequencyof positives for each group are shown in Table 2.

TABLE 2 Average microscopic lesion scores by treatment group and numberof animals with a positive microscopic score within groups. BronchoTreat- Pneumonia Broncho Bacterial Grp ment N lesion Necrosis PleuritisPneumonia Necrosis Pleuritis Colonies 1 Whole 15 1.07 0.67 1.67 53.33%40.00% 60.00% 20.00% Cell (8/15) (6/15) (9/15) (3/15) 2 Super- 15 0.870.8 1.13 46.67% 40.00% 46.67% 33.33% natant (7/15) (6/15) (7/15) (5/15)3 OMP 15 1.60 1.47^(c) 1.60^(c) 66.67% 60.00% 66.67% 46.67% (10/15) (9/15) (10/15)  (7/15) 4 APP 15 0.93 0.47^(c) 0.67^(b,c) 46.67% 26.67%46.67% 20.00% (7/15) (4/15) (7/15) (3/15) 5 Challenge 15 1.27^(a)0.93^(a) 1.93^(a,b)  66.67%^(a)  46.67%^(a)  66.67%^(a) 40.00% (10/15) (7/15) (10/15)  (6/15) 6 Strict 10 0.00^(a) 0.00^(a) 0.00^(a)  0.00%^(a)   0.00%^(a)   0.00%^(a)  0.00% Control (0/10) (0/10) (0/10)(0/10) ^(a)Groups 5 vs. 6 comparisons are statistically significantdifferent (p < 0.05, Wilcoxon Two Sample Test and Fishers Exact Testwhere appropriate). ^(b)Groups 4 vs. 5 comparisons are statisticallysignificant different (p < 0.05, Wilcoxon Two Sample Test and FishersExact Test where appropriate). ^(c)Groups 3 vs. 4 comparisons arestatistically significant different (p < 0.05, Wilcoxon Two Sample Testand Fishers Exact Test where appropriate).

Using the Wilcoxon Two-Sample Test, statistical analyses were run tocompare the means among treatment groups on average microscopic lunglesions scores. The Fisher Exact Test was also performed on the numberof positive scores for the percent positive animals per group for eachparameter. Statistical differences (p≦0.05) were found when comparingtreatment groups 5 (challenge) vs. 6 (strict control) forbronchopneumonia, necrosis, and pleuritis lesion scores, and percentpositive. The scores were zero in all parameters for treatment group 6(strict controls) (Table 2).

The highest bronchopneumonia lesion scores (1.60) were found intreatment group 3 (omp) followed by (1.27) treatment group 5(challenge). The lowest received score (0.87) was in group 2(supernatant) followed by (0.93) group 4 (APP). Scores forbronchopneumonia ranged from 0.87 to 1.60. Comparing necrosis lesionscores for treatment groups 1-5 ranged from 0.47 found in group 4 (APP)to 1.47 found in group 3 (omp). Statistical differences (p≦0.05) werefound when comparing treatment groups 3 vs. 4 for necrosis. Furthercomparisons of treatment groups 1-5 for the pleuritis parameter revealedlesion scores that ranged from 0.67 in group 4 (App) to 1.93 in group 5(challenge). Statistical differences (p≦0.05) were found when comparingtreatment groups 3 vs. 4 and 4 vs. 5 for pleuritis. Comparison oftreatment groups 1 vs. 4 was very close to being statistically different(p=0.0544).

Numerical comparisons of percent positives showed that when compared totreatment groups 1, 2, 4, and 5 treatment group 3 (omp) received thehighest scores for bronchopneumonia, necrosis, pleuritis, and bacterialcolonies of 66.67%, 60.00%, 66.67%, and 46.67% respectively. The highestscores were shared with treatment group 5 (challenge) forbronchopneumonia and pleuritis parameters. Furthermore, comparisons ofpercent positives showed that when compared to the treatment groups 1,2, 3, and 5 the lowest scores were in treatment group 4 (APP) forbronchopneumonia, necrosis, pleuritis, and bacterial colonies of 46.67%,26.67%, 46.67%, and 20.00% respectively. Treatment group 2 (supernatant)had the same lowest scores in bronchopneumonia and pleuritis parametersas group 4, and group 2 and group 1 (whole cell) had the same frequencyof bacterial colonies present. No statistical differences were found forpercent positives in the parameters listed in Table 2.

E. Secondary Efficacy Parameters

Secondary parameters were used to support primary efficacy parameters inthis study. Statistical analyses were performed on the followingsecondary parameters.

1. Clinical Observations

Observations for clinical signs were made from the day of vaccinationthroughout the study (days 0 through 41). Four main parameters werescored: body condition (gauntness) on a scale of 1 to 3 with 1 beingnormal and 3 being dead; behavior (depression); locomotion; andrespiration, each on a scale of 1 to 4 with 1 being normal and 4 beingdead. An average clinical score was used from all 4 parameters by whicha normal, healthy animal received a score of 1 while a severely affectedanimal (dead) could have a maximum score of 3.75. Table 3 shows theaverage scores by treatment group. Using the Wilcoxon Two-Sample Test,statistical analysis was only performed to compare groups 1 through 5 onaverage clinical observation scores.

TABLE 3 Daily average clinical scores for pre-challenge andpost-challenge periods within each treatment group. Pre- Post ChallengeChallenge Days Post Challenge Days Grp Treatment (0-35) Day 36 Day 37Day 38 Day 39 Day 40 Day 41 (36-41) 1 Whole Cell 1.001^(a,b) 1.25^(b)1.04 1.00 1.02 1.04 1.07 1.068^(a) 2 Supernatant 1.000^(a,b) 1.03 1.031.00 1.00^(a) 1.00^(a) 1.03^(a) 1.017^(a) 3 OMP 1.000^(a,b) 1.07^(b)1.03 1.05 1.00^(a) 1.05 1.13 1.058^(a) 4 APP 1.011^(b) 1.00^(a,b) 1.021.00 1.02 1.04 1.14 1.036^(a) 5 Challenge 1.013^(a) 1.12^(a) 1.08 1.121.28^(a) 1.14^(a) 1.14^(a) 1.148^(a) *6  Strict 1.000 1.00 1.00 1.001.00 1.00 1.00 1.000 *Treatment group 6 was not included in thestatistical analysis. ^(a)Groups 1-4 vs. 5 comparisons are statisticallysignificant different (p < 0.05, Wilcoxon Two Sample Test whereappropriate). ^(b)Groups 1-3 vs. 4 comparisons are statisticallysignificant different (p < 0.05, Wilcoxon Two Sample Test whereappropriate).

Comparison of the treatment groups 1-5 during the pre-challenge periodrevealed that during this time period compared to the treated groups 1-4the untreated challenge controls had the highest clinical scores of1.013. Treatment group 4 (APP) had the second highest mean clinicalscore of 1.011 followed by groups 1, 2 and 3 with scores of 1.001,1.000, and 1.000 respectively. Statistical differences (p≦0.05) werefound when comparing treatment groups 1-3 vs. group 5 from an overallmean score for the pre-challenge period. In addition statisticaldifferences (p≦0.05) were found when comparing groups 1-3 vs. group 4.No statistical differences were found when comparing group 4 vs. group5.

During the challenge period it was noted that treatment group 5(challenge) had the highest overall score of 1.148 followed by groups 1,3, 4, and 2 with scores of 1.068, 1.058, 1.036, and 1.017 respectively.For overall average clinical scores, statistical differences (p≦0.05)were found when comparing groups 1-4 vs. group 5. During the challengeperiod, treatment group 5 (challenge) had the highest scores on days 37through 41. Treatment group 1 (whole cell) had the highest score on day36. Furthermore, treatment group 1 (whole cell) scores fell numericallyfollowing day 36 spike from 1.25 to a range of 1.00 to 1.07. The lowestoverall score was in treatment group 2 (supernatant), and it wasstatistical different (p≦0.05) compared to group 5 (challenge) on days39, 40, and 41. Further comparison of group 4 (APP) showed that on day36, one day post challenge, there was a statistical difference (p≦0.05)compared to group 5 (challenge). Also, on day 36 groups 1 and 3 werestatistical different (p≦0.05) compared to group 4 (APP).

2. Rectal Temperatures

To monitor the effects of vaccination and challenge, rectal temperatureswere taken at different time points throughout the study (Days 0, 0+4hrs, 1, 3, 5, 7, 14, 21, 28, 34, 35, 36, 37, 38, 39, 40, and 41).Temperature spikes greater than (104.9° F.) were considered to besignificant. The number of animals with temperatures exceeding thecut-off are listed in Table 4. Using Fisher's Exact Test and ANOVA,statistical analyses were only performed to compare pyrexia scores forgroups 1 through 5.

Referring to FIG. 1 and Table 4 for the first vaccination time period ofday 0 to 21, there was a general spike in rectal temperature in alltreatment groups with a larger spike ≧104.9° F. at time 4 hours postvaccination in treatment groups 3 (omp) and 4 (APP) and continued to day1 for group 3 (omp). In group 1 (whole cell) there was a decrease intemperature on day 7 to 102° F., and statistical differences wereobserved (p≦0.05) when comparing group 1 vs. groups 2-5 on this day.Furthermore, the treated animal temperatures in groups 1-4 werenumerically less than the untreated groups 4-5 on day 7. On day 21 ofthe study the treated animals had numerically lower temperatures thanthe untreated and were found to be statistically different (p≦0.05) whencomparing groups 1-4 vs. group 5.

TABLE 4 Rectal temperatures (° F.) from day 0 to day 21 (firstvaccintion period). Day Grp Treatment Day 0 0 + 4 hrs. Day 1 Day 3 Day 5Day 7 Day 14 Day 21 1 Whole Cell 103.1 104.1^(b) 103.5^(b) 103.3 103.5102.0^(a,b) 103.4^(a,b) 103.7^(a,b) 2 Supernatant 103.2 104.2^(c)103.6^(c) 103.3 103.3 102.8^(a,b) 103.6 103.3^(a,b) 3 OMP 103.2104.9^(a,b,c,d) 104.9^(a,b,c) 103.3 103.3 102.8^(a,b) 103.6 103.5^(a) 4APP 103.3 105.1^(a,b,d) 103.9^(c) 103.2 103.5 102.9^(a,b) 103.8^(b)103.5^(a) 5 Challenge 103.1 103.8^(a) 103.5^(a) 103.5 103.6 103.6^(a)103.7^(a) 104.1^(a) 6 Strict 103.1 103.8 103.2 103.5 103.7 103.5 104.1104.1 *Treatment group 6 was not included in the statistical analysis.^(a)Groups 1-4 vs. 5 comparisons are statistically significant different(p < 0.05, ANOVA Test where appropriate). ^(b)Groups 1 vs. 2-4comparisons are statistically significant different (p < 0.05, ANOVAwhere appropriate). ^(c)Groups 2 vs. 3-4 comparisons are statisticallysignificant different (p < 0.05, ANOVA where appropriate). ^(d)Groups 3vs. 4 comparisons are statistically significant different (p < 0.05,ANOVA where appropriate).

Referring to FIG. 1 and Table 5, for the booster time period of day 22through day 35 it was observed that treatment group 4 (APP) had thehighest numerical rectal temperature on day 28 of the study. Statisticaldifferences were observed (p≦0.05) on day 28 when comparing treatmentgroups 1 and 2 vs. group 4.

TABLE 5 Rectal temperatures in degrees Fahrenheit (° F.) from day 22 today 35 (booster period). Group Treatment Day 28 Day 34 Day 35 1 WholeCell 103.7^(a) 103.5 103.9 2 Supernatant 103.6^(b) 103.7 104.0 3 OMP103.9 103.6 103.8 4 APP 104.3^(a,b) 103.5 103.7 5 Challenge 103.9 103.5103.7 6 Strict 103.9 103.5 103.7 *Treatment group 6 was not included inthe statistical analysis. ^(a)Groups 1 vs. 2-4 comparisons arestatistically significant different (p < 0.05, ANOVA where appropriate).^(b)Groups 2 vs. 3-4 comparisons are statistically significant different(p < 0.05, ANOVA where appropriate).

Referring to FIG. 1 and Table 6, for the challenge time period days 36through day 41 it was observed that treatment group 5 (challenge) hadthe highest numerical rectal temperatures on days 36, 37, 40, and 41;whereas, treatment group 1 (whole cell) had the highest rectaltemperatures on days 38 and 39. Furthermore, treatment group 4 (APP) hadthe lowest overall rectal temperatures on days 36, 37, 38, and 41. Inaddition treatment group 5 (challenge) had the same lowest temperatureon day 38 as treatment group 4, and groups 2 and 3 had the lowesttemperatures on days 39 and 40 respectively. Both groups 3 and 5 had aninitial rise in temperature one day post-challenge that then fell forgroup 3; however, the temperature for group 5 rose again on day 40.

TABLE 6 Rectal temperatures (° F.) from day 36 to day 41 (challengeperiod) and number of animals with pyrexia (≧104.90° F.) in parenthesesfor at least one day from day of challenge to necropsy. Group TreatmentDay 36 Day 37 Day 38 Day 39 Day 40 Day 41 % Present 1 Whole Cell 104.0104.1 103.9^(b) 104.0 103.7 103.8^(b) 53.33%^(b) (8/15) 2 Supernatant103.7^(a) 103.8^(a) 103.7 103.5 103.9 103.9^(c) 13.33%^(a,b) (2/15) 3OMP 104.3 103.9^(a) 103.8 103.8 103.4^(a) 103.8^(c) 33.33% (5/15) 4 APP103.5^(a) 103.6^(a) 103.5^(b) 103.7 103.6^(a) 103.1^(a,b,c) 7.14%^(a,b)(1/14) 5 Challenge 104.6^(a) 104.4^(a) 103.5 103.7 104.1^(a) 104.0^(a)66.67%^(a) (10/15) 6 Strict 103.7 103.8 103.7 103.6 103.6 103.5 103.7*Treatment group 6 and Spleen, Meninges, and Heart Blood were not partof the statistical analysis. ^(a)Groups 1-4 vs. 5 comparisons arestatistically significant different (p < 0.05, ANOVA and Fishers ExactTest where appropriate). ^(b)Groups 1 vs. 2-4 comparisons arestatistically significant different (p < 0.05, ANOVA and Fishers ExactTest where appropriate). ^(c)Groups 2-3 vs. 4 comparisons arestatistically significant different (p < 0.05, ANOVA and Fishers ExactTest where appropriate).

Further evaluation of the challenge period showed that the challengegroup 5 received the highest percent positive of animals that werefebrile with a value of 66.67%, which was statistically different(p≦0.05) when compared to groups 2 and 4. The second highest scorereceived was in treatment group 1 with a score of 53.33%, which was alsostatistically different (p≦0.05) when compared to groups 2 and 4. Groups4 (APP) and 2 (supernatant) received the lowest scores of 7.14% and13.33% respectively. Treatment group 3 (omp) had a score of 33.33%.

3. Bacterial Isolation

At necropsy, swabs of the nasal cavity, trachea, bronchi, meninges, andheart blood were collected for bacterial isolation. Sheep blood agarplates (5% with TSA) and MacConkey agar plates were inoculated with eachswab, streaked for isolation and incubated overnight at 37° C. Bloodagar plates were placed under anaerobic and aerobic conditions, and theMacConkey agar plates were placed under aerobic conditions only. Inaddition, chocolate agar plates were used only for culturing lungsamples and incubated at 37° C. under aerobic conditions. Fresh tissuesamples of the lung (3 lobes from sections containing lesions), liver,kidney, and tonsil were obtained. Swabs of each tissue were used toinoculate agar plates for bacterial isolation. Plates were incubatedalong with the swab samples mentioned above, and plates were observedfor presence of A. suis 24 hours post incubation. Biochemical analysiswas done on a random sample in each of the treatment groups to confirmthe presence of A. suis. Statistical analyses were only performed tocompare groups 1 through 5 on bacterial isolation scores using theFisher's Exact Test. The spleen, meninges, and heart blood were notstatistically analyzed. Tables 7a and 7b provides a summary of thebacteriology results.

TABLE 7a Number of percent positive A. suis animals in treatment groupsby bacterial isolation. Group Treatment Lung Liver Kidney Tonsil Nasal 1Whole Cell   40% 6.67% 6.67% 66.67% 13.33% (6/15) (1/15) (1/15) (10/15)(2/15) 2 Supernatant 33.33% 0.00% 13.33%  80.00% 13.33% (5/15) (0/15)(2/15) (12/15) (2/15) 3 OMP 33.33% 0.00% 0.00% 60.00%  6.67% a (5/15)(0/15) (0/15)  (9/15) (1/15) 4 APP 33.33% 6.67% 6.67% 60.00%  0.00% a(5/15) (1/15) (1/15)  (9/15) (0/15) 5 Challenge 73.33% 13.33%  20.00% 86.67% 46.67 a (11/15)  (2/15) (3/15) (13/15) (7/15) *6  Strict  0.00%0.00% 0.00%  0.00% 20.00% (0/10) (0/10) (0/10)  (0/10) (3/10) *Treatmentgroup 6 and Parameters Spleen, Meninges, and Heart Blood were not partof the statistical analysis. aGroups 1-4 vs. 5 comparisons arestatistically significant different (p < 0.05, Fishers Exact Test whereappropriate).

TABLE 7b Number of percent positive A. suis animals in treatment groupsby bacterial isolation. *Heart Group Treatment Trachea Bronchi *Spleen*Meninges blood 1 Whole Cell 6.67% 13.33% 0.00% 6.67% 6.67% (1/15)(2/15) (0/15) (1/15) (1/15) 2 Supernatant 6.67%  0.00% 0.00% 0.00% 0.00%(1/15) (0/15) (0/15) (0/15) (0/15) 3 OMP 6.67% 13.33% 6.67% 0.00% 0.00%(1/15) (2/15) (1/15) (0/15) (0/15) 4 APP 20.00%  20.00% 0.00% 6.67%0.00% (3/15) (3/15) (0/15) (1/15) (0/15) 5 Challenge 20.00%  26.67%6.67% 6.67% 6.67% (3/15) (4/15) (1/15) (1/15) (1/15) *6  Strict 6.67% 0.00% 0.00% 0.00% 0.00% (1/10) (0/10) (0/10) (0/10) (0/10) *Treatmentgroup 6 and Parameters Spleen, Meninges, and Heart Blood were not partof the statistical analysis. ^(a)Groups 1-4 vs. 5 comparisons arestatistically significant different (p < 0.05, Fishers Exact Test whereappropriate).

The recovery of A. suis in each of the target areas was the highest intreatment group 5 (challenge) compared to the other groups (1-4). Thehighest percentage of A. suis recovery was found in the tonsil followedby the lung. The recovery of A. suis was low in the spleen, meninges,and heart blood parameters with only a maximum recovery yield of 6.67%among the treatment groups. The strict controls, group 6, had three outof ten pigs positive for A. suis in nasal tissue, and one pig positivefor tracheal tissue. Statistical differences (p≦0.05) were found innasal tissue when comparing groups 3 (omp) and 4 (APP) vs. group 5(challenge).

Other common respiratory and bacterial organisms were isolated duringthe culturing process. Bordettella sp. was in high numbers in all groupsas well as alpha and beta streptococcal bacteria and Pasteurellamultocida. Other organisms isolated in very low numbers wereArcanobacterium pyrogenes, E. coli, Staphylococcus sp, Enterococcous sp,Proteus, blue fungii, and Pastunella sp.

4. Serology

Blood was drawn on study day 0, 7, 14, 21, 28, 34, and 41 of the study.Western blots were performed on pooled sera from treatment groups 1, 2,3, and 4 on days 0 and 34 of the study to measure immunoreactivity tothe fractions. Each treatment group consisted of three pooled groups,which consisted of 5 pigs from that treatment group. The western blotsshowed detectible seroconversion to the whole cell, supernatant, and ompfractions at 1:100 dilution, but not a 1:1000. The APP fraction had thebest reactivity and was detectable at 1:1000. For all groups, noreactivity was found in the day 0 samples prior to treatment.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by thefollowing claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Monteiro, Slavic, and Michael, 2000) J Clin Microbiol 38 (10):    3759-3762-   Oliveira et al, (2007) American Association Of Swine Veterinarians,    pp. 371-376.-   Oliveira S. (2007) Swine respiratory bacterial pathogens: bacterial    overview and vaccine trends. In: J Wiseman, M Varley, S McOrist & B    Kemp (Eds), Paradigms in Pig Science (pp. 196-178). Nottingham, UK:    Nottingham University Press.-   Rullo, Papp-Szabo and Michael, (2006) Biochimie et biologie    cellulaire 84 (2):184-90.-   Simpson (1949) Measurement of diversity. Nature 163:688-   Slavic, et al., (2000a) Can J Vet Res. 2000 April; 64 (2): 81-87.-   Slavic, et al., (2000b) J Clin Microbiol. 2000 October; 38 (10):    3759-3762.-   Versalovic et al, 1991, Nucleic Acids Research, Vol. 19, No. 24    6823-6831

What is claimed is:
 1. An immunogenic composition for reducing theincidence or severity of a clinical sign associated with Actinobacillussuis infection in a subject, comprising a) a supernatant collected fromone or more A. suis cultures grown to between 0.775 OD₆₀₀ and 0.815OD₆₀₀; and b) an adjuvant, wherein the reduction of the incidence of orthe severity of a clinical sign is relative to a subject not receivingthe immunogenic composition.
 2. The immunogenic composition of claim 1,wherein the supernatant is essentially free from A. suis cells.
 3. Theimmunogenic composition of claim 1, wherein the supernatant isinactivated.
 4. The immunogenic composition of claim 3, wherein thesupernatant is formalin inactivated.
 5. The immunogenic composition ofclaim 1, wherein the adjuvant is an oil-in-water emulsion anddimethyldioctadecylammonium bromide (DDA).
 6. The immunogeniccomposition of claim 1, wherein the subject is a porcine.
 7. A method ofprovoking an immune response against Actinobacillus suis in a subjectcomprising administering to the subject the immunogenic composition ofclaim
 1. 8. The method of claim 7, wherein the subject is a porcine. 9.A method of reducing the incidence of or severity of a clinical signassociated with Actinobacillus suis infection comprising the step ofadministering the immunogenic composition of claim 1 to a subject,wherein the reduction of the incidence of or the severity of a clinicalsign is relative to a subject not receiving the immunogenic composition.10. The method of claim 9, wherein the clinical sign is selected fromthe group consisting of meningitis, septicemia, metritis, pneumonia,crysipelas-like lesions, and abortion.
 11. The method of claim 9,wherein the subject is a porcine.
 12. A method of preparing theimmunogenic composition of claim 1 comprising: a) growing anActinobacillus suis culture to between 0.775 OD₆₀₀ and 0.815 OD₆₀₀; b)collecting a supernatant from the culture; c) filtering the supernatantto yield a filtrate; and d) mixing the filtrate with an adjuvant. 13.The method of claim 12, further comprising inactivating the supernatant.14. The method of claim 13, wherein the supernatant is inactivated priorto mixing with adjuvant.
 15. The method of claim 12, wherein theadjuvant is an oil-in-water emulsion and dimethyldioctadecylammoniumbromide (DDA).
 16. A kit comprising a) a filtered supernatant preparedby growing an Actinobacillus suis culture to between 0.775 OD₆₀₀ and0.815 OD₆₀₀, collecting a supernatant of the culture, and filtering thesupernatant; b) an adjuvant; and c) a container for packaging thesupernatant and adjuvant.
 17. The kit of claim 16, further comprisinginstructions for use of the kit.
 18. The kit of claim 16, furthercomprising a means of administering the filtered supernatant to asubject.